CN113406339A - Dry-type immunofluorescence quantitative method human Copeptin (CPP) detection kit - Google Patents
Dry-type immunofluorescence quantitative method human Copeptin (CPP) detection kit Download PDFInfo
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Abstract
The invention relates to a detection kit for detecting human Copeptin (CPP) by a dry immunofluorescence quantitative method, which comprises a box body, an upper cover and a reagent strip, wherein the reagent strip comprises a sample pad, a whole blood filter pad, a combination pad, a nitrocellulose membrane and absorbent paper, the nitrocellulose membrane is sequentially coated with a quality control C line and a detection T line, the quality control C line is arranged at one side close to the absorbent paper, the kit is marked by fluorescent microspheres, and a fluorescent antibody solid phase is arranged on the independent combination pad, so that the storage temperature requirement of a liquid phase antibody buffer solution is reduced; the invention selects chicken IgY and goat anti-chicken IgY paired antibodies as independent quality control line antibodies, has less cross with the mouse antibody, good specificity and stable quality control line signal value, and is beneficial to improving the accuracy and the repeatability of products; the blocking agent is added into the sample processing pad to reduce the influence of the interference antibody on the immunity detection to the maximum extent and avoid the false positive or false negative result of the detection.
Description
Technical Field
The invention relates to the field of in-vitro diagnosis immunological determination analysis, in particular to a human Copeptin (CPP) detection kit by a dry immunofluorescence assay.
Background
Copeptin (CPP), first discovered by Holwerda in 1972, was a leucine-rich glycopeptide derived from the C-terminus of the pro-AVP, consisting of 39 amino acids, with a relative molecular mass of 5000D. The pro-AVP consists of l64 amino acids and includes a signal peptide and 3 active fragments, vasopressin (AVP), pituitary hormone transporter II, and peptin (CPP). Early studies showed that copeptin may act as prolactin release factor, and recent studies suggest that copeptin may play a role in correcting during the structure formation of AVP precursors, and most likely interact with calnexin/calreticulum system to refold misfolded monomers, thereby stabilizing the biological effects of AVP. Researchers firstly found that the increase of the AVP level in vivo is obviously related to the prognosis of cardiovascular diseases such as angina pectoris, myocardial infarction and the like, but the in vitro measurement is quite difficult because the plasma half-life of the AVP is less than 30 min. The copeptin and AVP have a common precursor, the quantity of the copeptin is positively correlated with the quantity of the copeptin, the quantity of samples required for detecting the copeptin is small, the detection method is simple, convenient and quick, and has high stability, the stability of the copeptin in blood plasma or blood serum can reach more than 7 days at room temperature, and the copeptin can be maintained for 14 days at 4 ℃. Therefore, the copeptin can be used as a substitute for directly measuring the AVP concentration for clinical research and is expected to become a clinical prediction marker factor for the diseases.
The clinical application of copeptin detection mainly comprises the following aspects: 1. cerebrovascular disease; 2. cardiovascular diseases (such as heart failure, acute myocardial infarction, myocardial ischemia, etc.); 3. in other clinical aspects: (1) lower respiratory tract infections (including community-acquired pneumonia, chronic obstructive pneumonia in acute exacerbation phase, acute bronchitis, asthma exacerbation, etc.); (2) severe trauma associated infections (e.g., sepsis, systemic inflammatory response syndrome, hospitalized patients after cardiac surgery, etc.); (3) can be used for diagnosing diabetes insipidus.
To date, only four techniques have been used to determine copeptin concentrations: microtubule chemiluminescence, Radioimmunoassay (RIA), electrochemical assay, and enzyme-linked immunoassay (ELISA). Although their sensitivity can be satisfactory, the detection time is too long, so it is only used for scientific research and not clinically. Up to now, the point-of-care test (POCT) for copeptin has not been reported yet, and thus it largely limits the clinical value of copeptin.
Disclosure of Invention
The invention aims to solve the technical problem of filling the blank of human and peptide (CPP) immediate test in domestic markets and provides a dry immunofluorescence quantitative human and peptide (CPP) detection kit, wherein the kit is marked by fluorescent microspheres, and a fluorescent antibody solid phase is arranged on an independent binding pad, so that the storage temperature requirement of a liquid phase antibody buffer solution is reduced; meanwhile, the types of detection samples are increased, including serum, plasma and whole blood; the invention selects chicken IgY and goat anti-chicken IgY paired antibodies as independent quality control line antibodies, has less cross with the mouse antibody, good specificity and stable quality control line signal value, and is beneficial to improving the accuracy and the repeatability of products; the blocking agent is added into the sample processing pad to reduce the influence of the interference antibody on the immunity detection to the maximum extent and avoid the false positive or false negative result of the detection.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a dry immunofluorescence quantitative method human and peptide (CPP) detection kit comprises a box body, an upper cover and a reagent strip, wherein the box body and the upper cover are mutually connected in a buckling mode, a containing cavity is formed between the box body and the upper cover, the reagent strip is located in the containing cavity, the reagent strip comprises a sample pad, a whole blood filter pad, a combination pad, a nitrocellulose membrane and absorbent paper, the absorbent paper is located at one end of the nitrocellulose membrane, the combination pad is located at the other end of the nitrocellulose membrane, the whole blood filter pad is located at one end, far away from the nitrocellulose membrane, of the combination pad, the nitrocellulose membrane is sequentially coated with a quality control C line and a detection T line, the quality control C line is arranged on one side close to the absorbent paper, and the detection T line is arranged on one side close to the combination pad;
the upper cover is provided with a sample adding hole and a detection window, the sample adding hole is positioned above the sample pad, and the detection window is positioned above the detection T line and the quality control C line;
the quality control C line is coated with goat anti-chicken IgY antibody through antibody coating liquid, the concentration is 0.2-4mg/mL, and the antibody coating liquid of the quality control C line is prepared by 10mM PBS containing 0.5-5 wt% trehalose; the detection T line is prepared by coating a mouse-anti-human CPP monoclonal antibody with an antibody coating solution at a concentration of 0.5-3mg/mL, wherein the antibody coating solution of the detection T line is prepared by adding 0.5-5 wt% of sucrose into 10mM PBS.
As a further improvement of the technical scheme, the solid phase of the binding pad is provided with a fluorescent antibody, the fluorescent antibody comprises fluorescent microsphere solid phase liquid, 0.1-1mg/mL of mouse anti-human CPP monoclonal antibody marked by the fluorescent microsphere and 0.1-1mg/mL of chicken IgY, and the fluorescent microsphere solid phase liquid is prepared according to the conditions that 20mM, pH 8.0 and Tris contain 0.01-0.2 wt% of casein, 2-5 wt% of trehalose, 2-10 wt% of sucrose, 0.01-10 wt% of PVP and 0.01-5 wt% of Tween.
As a further improvement of the technical scheme, the combined pad needs to be pretreated by treatment liquid, and the treatment liquid contains macromolecular polymer and nonionic surfactant which are convenient for the fluorescent microspheres to rapidly release, protein for protecting the activity of the fluorescent microsphere antibodies and saccharide for increasing the activity of the labeled antibodies.
As a further improvement of the technical scheme, the treatment solution is 10mM PBS, the macromolecular polymer is 0.1-10 wt% PVP and 0.01-5 wt% PVA, the nonionic surfactant is 0.01-5 wt% Tween, the protein for protecting the activity of the fluorescent microsphere antibody is 0.01-10 wt% BSA, and the saccharide for increasing the activity of the labeled antibody is 1-10 wt% trehalose.
As a further improvement of the above technical solution, the method for labeling fluorescent microspheres comprises the following steps:
the microspheres are subjected to EDC/Sulfo-NHS coupled antibody in a two-step method, the microspheres are washed for 1 time by an activation buffer (50 mM MES buffer PH 6.0) before EDC and Sulfo-NHS are activated, and the microspheres are centrifuged and redissolved by the activation buffer; according to the microsphere: EDC: activating for 20-60min at a sulfoo-NHS mass ratio of 1:0.1-1:0.1-3, centrifuging, re-dissolving with an activation buffer solution, washing for 1 time, centrifuging, re-dissolving with a coupling buffer solution (50 mM MES buffer solution pH 6.5) with coupling of 0.1-1mg antibody per mg microsphere, performing ultrasonic treatment, and mixing and incubating for 2-3h by using a rotary disc mixer; quenching with 100 mM glycine, and incubating for 20-30 min in a rotary disc mixer; centrifuging, washing microspheres for 2 times by using a microsphere sealing buffer solution, centrifuging, re-dissolving by using the microsphere sealing buffer solution, and sealing for 2-4h by using a rotating disc type mixer; centrifuging, re-dissolving the microsphere storage solution, ultrasonically maintaining the microspheres in a monodisperse state, and storing at 4 ℃.
As a further improvement of the technical scheme, the microsphere blocking buffer solution is prepared according to 20mM and pH 8.0 Tris containing 0.2-5 wt% of casein; the microsphere storage solution is prepared according to 20mM, pH 8.0 and Tris containing 0.2-5 wt% of casein, 1-10 wt% of trehalose, 0.01-5 wt% of Tween and 0.05-0.5 wt% of sodium azide.
As a further improvement of the above technical solution, the method for preparing the fluorescent antibody solid phase comprises: diluting the marked fluorescent microspheres to 3-20 times by using fluorescent microsphere solid-phase liquid, spraying the fluorescent microspheres to the pretreated bonding pad according to the amount of 2-10uL/cm, and drying in a 37 ℃ oven.
As a further improvement of the technical scheme, the sample pad needs to be pretreated by the treatment solution, the treatment solution is selected from 10mM PBS containing 0.01-2mg/mL sealant, and the pretreatment method of the sample pad comprises the steps of putting the whole glass fiber into the pretreatment solution, completely soaking for 30min, then placing the glass fiber into a 37 ℃ oven for drying, and storing for later use.
As a further improvement of the technical scheme, the detection method of the dry immunofluorescence quantitative method human Copeptin (CPP) detection kit comprises the following steps: adding 100 mu L of sample to be detected into a sample adding hole, horizontally placing for 15 min at room temperature, combining the CPP in the sample to be detected and the mouse anti-human CPP monoclonal antibody marked by the fluorescent microsphere on the combination pad to form an antigen-antibody complex, moving forwards along the nitrocellulose membrane under the action of chromatography, and capturing by the mouse anti-human CPP monoclonal envelope antibody to form a complex when moving to a detection T line, wherein the fluorescent antibody signal of the complex is in direct proportion to the concentration of the CPP; the chicken IgY antibody marked by the fluorescent microspheres on the combination pad is combined with the goat anti-chicken IgY antibody to form a compound when continuously moving to the quality control C line; after being analyzed by a fluorescence immunoassay quantitative analyzer, the concentration of CPP in the sample can be calculated.
As a further improvement of the above technical solution, the method for manufacturing the sample to be tested comprises: 100 mu L of serum or plasma or whole blood sample is added into a single aliquot of detection buffer and mixed well, wherein the sample detection buffer is 20mM, and the pH is 8.0 Tris and contains 0.01-3 wt% PVP, 0.1-5 wt% S9, 0.1-5 wt% BSA and 0.1-0.5 v/v% PC300 for preparation.
By adopting the technical scheme, the invention has the following advantages:
1. the invention provides a rapid, accurate and dry immunofluorescence quantitative human peptide (CPP) detection kit, which adopts a novel fluorescent microsphere marking process, solves the problem that fluorescent microspheres are unstable and easy to aggregate in the marking process by optimizing the pH value of a buffer solution of a microsphere activation system and an antibody coupling system, and is more beneficial to the control of a later-stage production process; by adjusting the activation ratio of EDC and NHS, the problems of low antibody coupling efficiency and the like are solved, and the product detection sensitivity is improved; meanwhile, the fluorescent antibody is immobilized on the binding pad, so that the precision of the product is ensured on the basis of widening the storage condition of the kit.
2. The chicken IgY and goat anti-chicken IgY paired antibodies are selected as independent quality control line antibodies, the cross with the mouse source antibodies is less, the specificity is good, the quality control line signal value is stable, and the accuracy and the repeatability of the product are favorably improved.
3. The invention provides fluorescent microsphere solid-phase liquid, and the fluorescent microsphere solid phase is arranged on a combination pad, so that the problem of inconsistent storage conditions of a fluorescent marker and a kit is solved, the kit assembly process is simplified, and the production cost and time are reduced.
The invention is further illustrated by the following examples and figures.
Drawings
FIG. 1 is a schematic structural diagram of a dry immunofluorescence quantitative human Copeptin (CPP) detection kit according to the present invention.
Fig. 2 is a top view of fig. 1.
FIG. 3 is a schematic view of the structure of the reagent strip of the present invention.
In the figure, the position of the upper end of the main shaft,
1-box body, 2-upper cover, 3-sample pad, 4-whole blood filter pad, 5-combination pad, 6-nitrocellulose membrane, 7-absorbent paper, 8-detection T line, 9-quality control C line, 10-sample adding hole, 11-detection window and 12-reagent strip.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
as shown in figure 1, figure 2 and figure 3, a dry immunofluorescence quantitative human-peptide (CPP) detection kit comprises a box body 1, an upper cover 2 and a reagent strip 12, wherein the box body 1 and the upper cover 2 are mutually connected in a buckling manner, a containing cavity is formed between the box body 1 and the upper cover 2, the reagent strip 12 is positioned in the containing cavity, a sample adding hole 10 and a detection window 11 are arranged on the upper cover 2, the reagent strip 12 comprises a sample pad 3, a whole blood filter pad 4, a combination pad 5, a nitrocellulose membrane 6 and absorbent paper 7, the absorbent paper 7 is positioned at one end of the nitrocellulose membrane 6, the combination pad 5 is positioned at the other end of the nitrocellulose membrane 6, the whole blood filter pad 4 is positioned at one end of the combination pad 5, which is far away from the nitrocellulose membrane 6, the combination pad 5 and the whole blood filter pad 4, the sample pad 3 is positioned at one end of the whole blood filter pad 4, and the nitrocellulose membrane 6, the combination pad 5 and the whole blood filter pad 4 are positioned at one end of the combination pad 5, The sample pad 3 and the absorbent paper 7 are sequentially adhered to the PVC board to assemble the reagent strip 12.
The nitrocellulose membrane 6 is coated with a quality control C line 9 and a detection T line 8 in sequence, the quality control C line 9 is arranged on one side close to the absorbent paper 7, the detection T line 8 is arranged on one side close to the combination pad 5, the sample adding hole 10 is arranged above the sample pad 3, and the detection window 11 is arranged above the detection T line 8 and the quality control C line 9.
The quality control C line 9 is coated with goat anti-chicken IgY antibody through antibody coating liquid, the concentration is 1mg/mL, and the antibody coating liquid of the quality control C line 9 is prepared by 10mM PBS containing 2 wt% trehalose; the detection T line 8 is prepared by coating a mouse anti-human CPP monoclonal antibody with an antibody coating solution, wherein the concentration of the antibody coating solution is 1.5mg/mL, and the antibody coating solution of the detection T line 8 is 10mM PBS containing 3 wt% of sucrose.
The solid phase of the binding pad 5 is provided with a fluorescent antibody, the fluorescent antibody comprises fluorescent microsphere solid phase liquid, a mouse anti-human CPP monoclonal antibody of 0.5mg/mL marked by the fluorescent microsphere and chicken IgY of 0.3mg/mL, the fluorescent microsphere solid phase liquid is prepared according to the condition that 20mM, pH 8.0 Tris contain 0.1 wt% casein, 5 wt% trehalose, 5 wt% sucrose, 0.05 wt% PVP and 0.02 wt% Tween, and the binding pad 5 is positioned between the whole blood filter pad 4 and the nitrocellulose membrane 6 and is overlapped with the whole blood filter pad 4 and the nitrocellulose membrane 6 in a cross mode by 1-2 mM.
The combination pad 5 needs to be pretreated by treatment liquid, and the treatment liquid contains macromolecular polymer and nonionic surfactant which are convenient for the fluorescent microspheres to rapidly release, protein for protecting the activity of the fluorescent microsphere antibody and saccharide for increasing the activity of the labeled antibody. The treatment fluid is 10mM PBS, the macromolecular polymer is 0.1 wt% PVP, 0.05 wt% PVA, the nonionic surfactant is 0.05 wt% Tween, the activity of the fluorescent microsphere antibody is protected, 1 wt% BSA is achieved, and the activity of the labeled antibody is increased by 3 wt% trehalose.
The method for labeling the fluorescent microspheres comprises the following steps:
the microspheres were conjugated with two-step EDC/Sulfo-NHS. Microspheres were washed 1 time with activation buffer (50 mM MES buffer pH 6.0) before EDC and Sulfo-NHS activation, centrifuged, and reconstituted with activation buffer. According to the microsphere: EDC: activating for 30min at a Sulfo-NHS mass ratio of 1:0.2:0.6, centrifuging, re-dissolving in an activation buffer solution, washing for 1 time, centrifuging, re-dissolving in a coupling buffer solution (50 mM MES buffer solution, pH 6.5), coupling 0.1mg antibody per mg microsphere, performing ultrasonic treatment, and mixing and incubating for 2h by using a rotating disc mixer. Quench with final concentration 100 mM glycine and incubate with mixing in a rotating disk mixer for 30 min. Centrifuging, washing microspheres for 2 times by microsphere sealing buffer solution, centrifuging, re-dissolving by microsphere sealing buffer solution, and sealing for 2h by a rotating disc type mixer. Centrifuging, re-dissolving the microsphere storage solution, ultrasonically maintaining the microspheres in a monodisperse state, and storing at 4 ℃.
The microsphere blocking buffer was prepared at 20mM, pH 8.0 Tris with 2 wt% casein.
The microsphere storage solution was prepared at 20mM, pH 8.0 Tris containing 0.5 wt% casein, 3 wt% trehalose, 0.02 wt% Tween and 0.1 wt% sodium azide.
The method for the fluorescent antibody solid phase comprises the following steps: diluting the marked fluorescent microspheres to 8 times by using solid phase liquid, spraying the diluted fluorescent microspheres to the pretreated combination pad 5 according to the amount of 5uL/cm, and drying in an oven at 37 ℃.
The whole blood filter pad 4 is preferably excellent in performance in all aspects, can quickly separate blood samples in a short time, can greatly inhibit hemolysis, has high stability and uniformity, does not need to be processed, and can be directly used.
The sample pad 3 needs to be pretreated by treatment fluid, the formula of the treatment fluid is that 10mM PBS contains 0.5mg/mL sealant, the sealant can actively neutralize interference antibodies (human anti-animal species antibodies, human anti-mouse antibodies, RF, heterophilic antibodies and the like), and the pretreatment method of the sample pad 3 is that the whole glass fiber is placed in the treatment fluid, completely soaked for 30min, then placed in a 37 ℃ oven for drying and stored for later use.
The final concentration of the sealant is selected from 0.1mg/mL, 0.3mg/mL, 0.5mg/mL and 0.8mg/mL, and the sealing effect of 0.5mg/mL and 0.8mg/mL meets the requirement of a false positive sample which does not accord with the clinical result after the test; through quality control product tests, 0.1mg/mL, 0.3mg/mL and 0.5mg/mL can meet the requirements; by combining the 2 test results, the sealant with the final concentration of 0.5mg/mL can meet the product requirements.
The blocking agent is added into the sample processing pad 3, and the blocking agent with good performance in all aspects is screened out by strictly screening and repeatedly verifying various active blocking type blocking agents, passive adsorption type blocking agents, blocking agents and other raw materials, so that the influence of interference antibodies on immunoassay is reduced to the maximum extent, and false positive or false negative of detection results is avoided.
A detection method of a dry immunofluorescence quantitative human Copeptin (CPP) detection kit comprises the following steps: adding 100 mu L of sample to be detected into a sample adding hole 10, horizontally placing for 15 min at room temperature, combining the CPP in the sample to be detected and the mouse anti-human CPP monoclonal antibody marked by the fluorescent microspheres on the combination pad 5 to form an antigen-antibody complex, moving forwards along the nitrocellulose membrane 6 under the action of chromatography, and capturing by the mouse anti-human CPP monoclonal envelope antibody to form a complex when moving to a detection T line 8, wherein the fluorescent antibody signal of the complex is in direct proportion to the CPP concentration; the chicken IgY antibody marked by the fluorescent microspheres on the binding pad 5 continuously migrates to the quality control C line 9 and is combined with the goat anti-chicken IgY antibody to form a compound, and the concentration of CPP in the sample can be calculated after the analysis of a fluorescence immunoassay quantitative analyzer.
The manufacturing method of the sample to be detected comprises the following steps: 100 μ L of serum or plasma or whole blood samples were added to a single aliquot of assay buffer, mixed well, and formulated in 20mM assay buffer, pH 8.0 Tris solution containing 0.5 wt% PVP, 0.2 wt% S9, 2 wt% BSA, and 0.5 v/v% PC 300.
Example 2:
as shown in figure 1, figure 2 and figure 3, a dry immunofluorescence quantitative human-peptide (CPP) detection kit comprises a box body 1, an upper cover 2 and a reagent strip 12, wherein the box body 1 and the upper cover 2 are mutually connected in a buckling manner, a containing cavity is formed between the box body 1 and the upper cover 2, the reagent strip 12 is positioned in the containing cavity, a sample adding hole 10 and a detection window 11 are arranged on the upper cover 2, the reagent strip 12 comprises a sample pad 3, a whole blood filter pad 4, a combination pad 5, a nitrocellulose membrane 6 and absorbent paper 7, the absorbent paper 7 is positioned at one end of the nitrocellulose membrane 6, the combination pad 5 is positioned at the other end of the nitrocellulose membrane 6, the whole blood filter pad 4 is positioned at one end of the combination pad 5, which is far away from the nitrocellulose membrane 6, the combination pad 5 and the whole blood filter pad 4, the sample pad 3 is positioned at one end of the whole blood filter pad 4, and the nitrocellulose membrane 6, the combination pad 5 and the whole blood filter pad 4 are positioned at one end of the combination pad 5, The sample pad 3 and the absorbent paper 7 are sequentially adhered to the PVC board to assemble the reagent strip 12. The nitrocellulose membrane 6 is coated with a quality control C line 9 and a detection T line 8 in sequence, the quality control C line 9 is arranged on one side close to the absorbent paper 7, the detection T line 8 is arranged on one side close to the combination pad 5, the sample adding hole 10 is arranged above the sample pad 3, and the detection window 11 is arranged above the detection T line 8 and the quality control C line 9.
The quality control C line 9 is coated with goat anti-chicken IgY antibody through antibody coating liquid, the concentration is 0.2mg/mL, and the antibody coating liquid of the quality control C line 9 is prepared by 10mMPBS containing 0.5 wt% of trehalose; the detection T line 8 is prepared by coating a mouse anti-human CPP monoclonal antibody with an antibody coating solution, wherein the concentration of the antibody coating solution is 0.5mg/mL, and the antibody coating solution of the detection T line 8 is 10mM PBS containing 0.5 wt% of sucrose.
The solid phase of the binding pad 5 is provided with a fluorescent antibody, and the fluorescent antibody comprises fluorescent microsphere solid phase liquid, a mouse anti-human CPP monoclonal antibody of 0.1mg/mL and chicken IgY of 0.1mg/mL marked by the fluorescent microsphere. The fluorescent microsphere solid phase solution is prepared according to 20mM, pH 8.0 and Tris containing 0.01 wt% of casein, 2 wt% of trehalose, 2 wt% of sucrose, 0.01 wt% of PVP and 0.01 wt% of Tween. The conjugate pad 5 is located between the whole blood filter pad 4 and the nitrocellulose membrane 6 and overlaps both across by 1-2 mm.
The combination pad 5 needs to be pretreated by treatment liquid, and the treatment liquid contains macromolecular polymer and nonionic surfactant which are convenient for the fluorescent microspheres to rapidly release, protein for protecting the activity of the fluorescent microsphere antibody and saccharide for increasing the activity of the labeled antibody. The treatment fluid is 10mM PBS containing 0.1 wt% PVP, 0.01 wt% PVA, 0.01 wt% Tween, 0.01 wt% BSA, 1 wt% trehalose.
The method for labeling the fluorescent microspheres comprises the following steps:
the microspheres were conjugated with two-step EDC/Sulfo-NHS. Microspheres were washed 1 time with activation buffer (50 mM MES buffer pH 6.0) before EDC and Sulfo-NHS activation, centrifuged, and reconstituted with activation buffer. According to the microsphere: EDC: activating for 20min at a sulfoo-NHS mass ratio of 1:0.1:0.1, centrifuging, re-dissolving in an activation buffer, washing for 1 time, centrifuging, re-dissolving in a coupling buffer (50 mM MES buffer pH 6.5), coupling 0.1mg antibody per mg microsphere, performing ultrasonic treatment, and mixing and incubating for 2h by using a rotary disc mixer. Quench with final concentration 100 mM glycine and incubate with mixing in a rotating disk mixer for 20 min. Centrifuging, washing microspheres for 2 times by microsphere sealing buffer solution, centrifuging, re-dissolving by microsphere sealing buffer solution, and sealing for 2h by a rotating disc type mixer. Centrifuging, re-dissolving the microsphere storage solution, ultrasonically maintaining the microspheres in a monodisperse state, and storing at 4 ℃.
The microsphere blocking buffer was prepared at 20mM, pH 8.0 Tris, with 0.2 wt% casein.
The microsphere preservation solution is prepared according to 20mM, pH 8.0 and Tris, wherein the microsphere preservation solution contains 0.2 wt% of casein, 1 wt% of trehalose, 0.01 wt% of Tween and 0.05 wt% of sodium azide.
The method for the fluorescent antibody solid phase comprises the following steps: diluting the marked fluorescent microspheres to 3 times by using solid phase liquid, spraying the diluted fluorescent microspheres to the pretreated combination pad 5 according to the amount of 2uL/cm, and drying in an oven at 37 ℃.
The whole blood filter pad 4 is preferably excellent in performance in all aspects, can quickly separate blood samples in a short time, can greatly inhibit hemolysis, has high stability and uniformity, does not need to be processed, and can be directly used.
The sample pad 3 needs to be pretreated by treatment fluid, the formula of the treatment fluid is that 10mM PBS contains 0.01mg/mL sealant, the sealant can actively neutralize interference antibodies (human anti-animal species antibodies, human anti-mouse antibodies, RF, heterophilic antibodies and the like), and the pretreatment method of the sample pad 3 is that the whole glass fiber is placed in the treatment fluid, completely soaked for 30min, then placed in a 37 ℃ oven for drying and stored for later use.
A detection method of a dry immunofluorescence quantitative human Copeptin (CPP) detection kit comprises the following steps: adding 100 mu L of sample to be detected into a sample adding hole 10, horizontally placing for 15 min at room temperature, combining the CPP in the sample to be detected and the mouse anti-human CPP monoclonal antibody marked by the fluorescent microspheres on the combination pad 5 to form an antigen-antibody complex, moving forwards along the nitrocellulose membrane 6 under the action of chromatography, and capturing by the mouse anti-human CPP monoclonal envelope antibody to form a complex when moving to a detection T line 8, wherein the fluorescent antibody signal of the complex is in direct proportion to the CPP concentration; the chicken IgY antibody marked by the fluorescent microspheres on the binding pad 5 continuously migrates to the quality control C line 9 and is combined with the goat anti-chicken IgY antibody to form a compound, and the concentration of CPP in the sample can be calculated after the analysis of a fluorescence immunoassay quantitative analyzer.
The manufacturing method of the sample to be detected comprises the following steps: 100 μ L of serum or plasma or whole blood samples were added to a single aliquot of assay buffer, mixed well, and formulated in 20mM assay buffer, pH 8.0 Tris solution containing 0.01 wt% PVP, 0.1 wt% S9, 0.1 wt% BSA, and 0.1 v/v% PC 300.
Example 3:
as shown in figure 1, figure 2 and figure 3, a dry immunofluorescence quantitative human-peptide (CPP) detection kit comprises a box body 1, an upper cover 2 and a reagent strip 12, wherein the box body 1 and the upper cover 2 are mutually connected in a buckling manner, a containing cavity is formed between the box body 1 and the upper cover 2, the reagent strip 12 is positioned in the containing cavity, a sample adding hole 10 and a detection window 11 are arranged on the upper cover 2, the reagent strip 12 comprises a sample pad 3, a whole blood filter pad 4, a combination pad 5, a nitrocellulose membrane 6 and absorbent paper 7, the absorbent paper 7 is positioned at one end of the nitrocellulose membrane 6, the combination pad 5 is positioned at the other end of the nitrocellulose membrane 6, the whole blood filter pad 4 is positioned at one end of the combination pad 5, which is far away from the nitrocellulose membrane 6, the combination pad 5 and the whole blood filter pad 4, the sample pad 3 is positioned at one end of the whole blood filter pad 4, and the nitrocellulose membrane 6, the combination pad 5 and the whole blood filter pad 4 are positioned at one end of the combination pad 5, The sample pad 3 and the absorbent paper 7 are sequentially adhered to the PVC board to assemble the reagent strip 12. The nitrocellulose membrane 6 is coated with a quality control C line 9 and a detection T line 8 in sequence, the quality control C line 9 is arranged on one side close to the absorbent paper 7, the detection T line 8 is arranged on one side close to the combination pad 5, the sample adding hole 10 is arranged above the sample pad 3, and the detection window 11 is arranged above the detection T line 8 and the quality control C line 9.
The quality control C line 9 is coated with goat anti-chicken IgY antibody through antibody coating liquid, the concentration is 4mg/mL, and the antibody coating liquid of the quality control C line 9 is prepared by 10mMPBS containing 5 wt% of trehalose; the detection T line 8 is prepared by coating a mouse anti-human CPP monoclonal antibody with an antibody coating solution, wherein the concentration of the antibody coating solution is 3mg/mL, and the antibody coating solution of the detection T line 8 is 10mM PBS containing 5 wt% of sucrose.
The solid phase of the binding pad 5 is provided with a fluorescent antibody, and the fluorescent antibody comprises fluorescent microsphere solid phase liquid, 1mg/mL of mouse anti-human CPP monoclonal antibody marked by the fluorescent microsphere and 1mg/mL of chicken IgY. The fluorescent microsphere solid phase solution is prepared according to 20mM, pH 8.0 Tris containing 0.2 wt% casein, 5 wt% trehalose, 10 wt% sucrose, 10 wt% PVP and 5 wt% Tween. The conjugate pad 5 is located between the whole blood filter pad 4 and the nitrocellulose membrane 6 and overlaps both across by 1-2 mm.
The combination pad 5 needs to be pretreated by treatment liquid, and the treatment liquid contains macromolecular polymer and nonionic surfactant which are convenient for the fluorescent microspheres to rapidly release, protein for protecting the activity of the fluorescent microsphere antibody and saccharide for increasing the activity of the labeled antibody. The treatment fluid is 10mM PBS containing 10 wt% PVP, 5 wt% PVA, 5 wt% Tween, 10 wt% BSA, 10 wt% trehalose.
The method for labeling the fluorescent microspheres comprises the following steps:
the microspheres were conjugated with two-step EDC/Sulfo-NHS. Microspheres were washed 1 time with activation buffer (50 mM MES buffer pH 6.0) before EDC and Sulfo-NHS activation, centrifuged, and reconstituted with activation buffer. According to the microsphere: EDC: activating for 60min at a Sulfo-NHS mass ratio of 1:1:3, centrifuging, re-dissolving in an activation buffer solution, washing for 1 time, centrifuging, re-dissolving in a coupling buffer solution (50 mM MES buffer solution pH 6.5), coupling 1mg of antibody per mg of microsphere, performing ultrasonic treatment, and mixing and incubating for 3h by using a rotary disc mixer. Quench with final concentration 100 mM glycine and incubate with mixing in a rotating disk mixer for 30 min. Centrifuging, washing microspheres for 2 times by microsphere sealing buffer solution, centrifuging, re-dissolving by microsphere sealing buffer solution, and sealing for 4h by a rotating disc type mixer. Centrifuging, re-dissolving the microsphere storage solution, ultrasonically maintaining the microspheres in a monodisperse state, and storing at 4 ℃.
The microsphere blocking buffer was prepared at 20mM, pH 8.0 Tris, with 5 wt% casein.
The microsphere preservation solution is prepared according to 20mM, pH 8.0 Tris, 5 wt% of casein, 10 wt% of trehalose, 5 wt% of Tween and 0.5 wt% of sodium azide.
The method for the fluorescent antibody solid phase comprises the following steps: diluting the marked fluorescent microspheres to 20 times by using solid phase liquid, spraying the diluted fluorescent microspheres to the pretreated combination pad 5 according to the amount of 10uL/cm, and drying in an oven at 37 ℃.
The whole blood filter pad 4 is preferably excellent in performance in all aspects, can quickly separate blood samples in a short time, can greatly inhibit hemolysis, has high stability and uniformity, does not need to be processed, and can be directly used.
The sample pad 3 needs to be pretreated by treatment liquid, the formula of the treatment liquid is that 10mM PBS contains 2mg/mL sealant, the sealant can actively neutralize interference antibodies (human anti-animal species antibodies, human anti-mouse antibodies, RF, heterophilic antibodies and the like), and the pretreatment method of the sample pad 3 is that the whole glass fiber is placed in the treatment liquid, completely soaked for 30min, then placed in a 37 ℃ oven for drying and stored for later use.
A detection method of a dry immunofluorescence quantitative human Copeptin (CPP) detection kit comprises the following steps: adding 100 mu L of sample to be detected into a sample adding hole 10, horizontally placing for 15 min at room temperature, combining the CPP in the sample to be detected and the mouse anti-human CPP monoclonal antibody marked by the fluorescent microspheres on the combination pad 5 to form an antigen-antibody complex, moving forwards along the nitrocellulose membrane 6 under the action of chromatography, and capturing by the mouse anti-human CPP monoclonal envelope antibody to form a complex when moving to a detection T line 8, wherein the fluorescent antibody signal of the complex is in direct proportion to the CPP concentration; the chicken IgY antibody marked by the fluorescent microspheres on the binding pad 5 continuously migrates to the quality control C line 9 and is combined with the goat anti-chicken IgY antibody to form a compound, and the concentration of CPP in the sample can be calculated after the analysis of a fluorescence immunoassay quantitative analyzer.
The manufacturing method of the sample to be detected comprises the following steps: 100 μ L of serum or plasma or whole blood samples were added to a single aliquot of assay buffer, mixed well, and formulated in 20mM assay buffer, pH 8.0 Tris solution containing 3 wt% PVP, 5 wt% S9, 5 wt% BSA, and 0.5 v/v% PC 300.
Example 4:
as shown in figure 1, figure 2 and figure 3, a dry immunofluorescence quantitative human-peptide (CPP) detection kit comprises a box body 1, an upper cover 2 and a reagent strip 12, wherein the box body 1 and the upper cover 2 are mutually connected in a buckling manner, a containing cavity is formed between the box body 1 and the upper cover 2, the reagent strip 12 is positioned in the containing cavity, a sample adding hole 10 and a detection window 11 are arranged on the upper cover 2, the reagent strip 12 comprises a sample pad 3, a whole blood filter pad 4, a combination pad 5, a nitrocellulose membrane 6 and absorbent paper 7, the absorbent paper 7 is positioned at one end of the nitrocellulose membrane 6, the combination pad 5 is positioned at the other end of the nitrocellulose membrane 6, the whole blood filter pad 4 is positioned at one end of the combination pad 5, which is far away from the nitrocellulose membrane 6, the combination pad 5 and the whole blood filter pad 4, the sample pad 3 is positioned at one end of the whole blood filter pad 4, and the nitrocellulose membrane 6, the combination pad 5 and the whole blood filter pad 4 are positioned at one end of the combination pad 5, The sample pad 3 and the absorbent paper 7 are sequentially adhered to the PVC board to assemble the reagent strip 12.
The nitrocellulose membrane 6 is coated with a quality control C line 9 and a detection T line 8 in sequence, the quality control C line 9 is arranged on one side close to the absorbent paper 7, the detection T line 8 is arranged on one side close to the combination pad 5, the sample adding hole 10 is arranged above the sample pad 3, and the detection window 11 is arranged above the detection T line 8 and the quality control C line 9.
The quality control C line 9 is coated with goat anti-chicken IgY antibody through antibody coating liquid, the concentration is 2mg/mL, and the antibody coating liquid of the quality control C line 9 is prepared by 10mM PBS containing 2.5 wt% trehalose; the detection T line 8 is prepared by coating a mouse anti-human CPP monoclonal antibody with an antibody coating solution, wherein the concentration of the antibody coating solution is 1.5mg/mL, and the antibody coating solution of the detection T line 8 is 10mM PBS containing 2.5 wt% of sucrose.
The solid phase of the binding pad 5 is provided with a fluorescent antibody, the fluorescent antibody comprises fluorescent microsphere solid phase liquid, a mouse anti-human CPP monoclonal antibody of 0.5mg/mL marked by the fluorescent microsphere and chicken IgY of 0.5mg/mL, the fluorescent microsphere solid phase liquid is prepared according to the condition that 20mM, pH 8.0 Tris contain 0.1 wt% casein, 3.5 wt% trehalose, 6 wt% sucrose, 5 wt% PVP and 2.5 wt% Tween, and the binding pad 5 is positioned between the whole blood filter pad 4 and the nitrocellulose membrane 6 and is overlapped with the whole blood filter pad 4 and the nitrocellulose membrane 6 in a cross mode by 1-2 mM.
The combination pad 5 needs to be pretreated by treatment liquid, and the treatment liquid contains macromolecular polymer and nonionic surfactant which are convenient for the fluorescent microspheres to rapidly release, protein for protecting the activity of the fluorescent microsphere antibody and saccharide for increasing the activity of the labeled antibody. The treatment fluid is 10mM PBS, the macromolecular polymer is 5 wt% PVP, 2.5 wt% PVA, the nonionic surfactant is 2.5 wt% Tween, the activity of the fluorescent microsphere antibody is protected by 5 wt% BSA, and the activity of the labeled antibody is increased by 5.5 wt% trehalose.
The method for labeling the fluorescent microspheres comprises the following steps:
the microspheres were conjugated with two-step EDC/Sulfo-NHS. Microspheres were washed 1 time with activation buffer (50 mM MES buffer pH 6.0) before EDC and Sulfo-NHS activation, centrifuged, and reconstituted with activation buffer. According to the microsphere: EDC: activating for 40min at a sulfoo-NHS mass ratio of 1:0.5:1.5, centrifuging, re-dissolving in an activation buffer, washing for 1 time, centrifuging, re-dissolving in a coupling buffer (50 mM MES buffer pH 6.5), coupling 0.5mg antibody per mg microsphere, performing ultrasonic treatment, and mixing and incubating for 2.5h by using a rotary disc mixer. Quench with final concentration 100 mM glycine and incubate with mixing in a rotating disk mixer for 25 min. Centrifuging, washing microspheres for 2 times by microsphere sealing buffer solution, centrifuging, re-dissolving by microsphere sealing buffer solution, and sealing for 3h by a rotating disc type mixer. Centrifuging, re-dissolving the microsphere storage solution, ultrasonically maintaining the microspheres in a monodisperse state, and storing at 4 ℃.
The microsphere blocking buffer was prepared at 20mM, pH 8.0 Tris, 2.5 wt% casein.
The microsphere storage solution was prepared from 20mM, pH 8.0 Tris containing 2.5 wt% casein, 5.5 wt% trehalose, 2.5 wt% Tween and 0.25 wt% sodium azide.
The method for the fluorescent antibody solid phase comprises the following steps: diluting the marked fluorescent microspheres to 11 times by using solid phase liquid, spraying the diluted fluorescent microspheres to the pretreated combination pad 5 according to the amount of 6uL/cm, and drying in an oven at 37 ℃.
The whole blood filter pad 4 is preferably excellent in performance in all aspects, can quickly separate blood samples in a short time, can greatly inhibit hemolysis, has high stability and uniformity, does not need to be processed, and can be directly used.
The sample pad 3 needs to be pretreated by treatment liquid, the formula of the treatment liquid is that 1mg/mL sealant is contained in 10mM PBS, the sealant can actively neutralize interference antibodies (human anti-animal species antibodies, human anti-mouse antibodies, RF, heterophilic antibodies and the like), and the pretreatment method of the sample pad 3 comprises the steps of putting the whole glass fiber into the treatment liquid, completely soaking for 30min, then placing the glass fiber into a 37 ℃ oven for drying, and storing for later use.
The blocking agent is added into the sample processing pad 3, and the blocking agent with good performance in all aspects is screened out by strictly screening and repeatedly verifying various active blocking type blocking agents, passive adsorption type blocking agents, blocking agents and other raw materials, so that the influence of interference antibodies on immunoassay is reduced to the maximum extent, and false positive or false negative of detection results is avoided.
A detection method of a dry immunofluorescence quantitative human Copeptin (CPP) detection kit comprises the following steps: adding 100 mu L of sample to be detected into a sample adding hole 10, horizontally placing for 15 min at room temperature, combining the CPP in the sample to be detected and the mouse anti-human CPP monoclonal antibody marked by the fluorescent microspheres on the combination pad 5 to form an antigen-antibody complex, moving forwards along the nitrocellulose membrane 6 under the action of chromatography, and capturing by the mouse anti-human CPP monoclonal envelope antibody to form a complex when moving to a detection T line 8, wherein the fluorescent antibody signal of the complex is in direct proportion to the CPP concentration; the chicken IgY antibody marked by the fluorescent microspheres on the binding pad 5 continuously migrates to the quality control C line 9 and is combined with the goat anti-chicken IgY antibody to form a compound, and the concentration of CPP in the sample can be calculated after the analysis of a fluorescence immunoassay quantitative analyzer.
The manufacturing method of the sample to be detected comprises the following steps: 100 μ L of serum or plasma or whole blood samples were added to a single aliquot of assay buffer, mixed well, and prepared with 20mM assay buffer, pH 8.0 Tris solution containing 1.5 wt% PVP, 2.5 wt% S9, 2.5 wt% BSA, and 0.3 v/v% PC 300.
Comparative example 1
The same procedure as in example 1, except that the formulation of the assay buffer was different, the assay buffer in example 1 was a 20mM Tris solution containing 0.5 wt% PVP, 0.2 wt% S9, 2 wt% BSA, and 0.5 v/v% PC 300; the detection buffer in comparative example 1 was prepared as a 50mM Tris solution containing 0.2 wt% PVP, 0.5 wt% PEG, 5 wt% BSA, 0.05 wt% casein and 0.5 v/v% PC 300.
The detection buffer solution is placed at 37 ℃ for destructive test, the stability of the detection buffer solution is inspected, and the detection standards are as follows:
the appearance of the detection buffer solution is colorless and transparent, and the color development degree is uniform and has no difference; the accuracy of the detection buffer should be satisfactory.
The test results are as follows:
table 1 results of measuring stability of buffer physical state
TABLE 2 detection buffer solution 37 ℃ accelerated destruction followed by the detection results of quality control articles (1-3) (unit: pmol/L)
TABLE 3 detection buffer solution 37 ℃ accelerated destruction followed by the quality control (4-6) test results (unit: pmol/L)
According to the experimental principle of stability, the formula of arrhenius: d (Ink)/dT = Ea/RT2 Ea, and is stored at room temperature for 24 months, which corresponds to a destruction at 37 ℃ for 180 days.
Tests show that the detection buffer solution of the embodiment has a long storage life and can be stabilized for at least 24 months, the color, the property and the performance of the quality control product of the sample buffer solution are better than those of the detection buffer solution of the comparative example 1, and the results are more consistent with those of the kit on the market, so that the detection buffer solution can be used as a better choice of the kit.
Comparative examples 2 to 4
The other operations are the same as those in example 1, the only difference is that the formula for detecting the T-line 8 CPP monoclonal antibody coating is different, and the preparation methods of the antibody coating liquid are respectively as follows:
comparative example 2, preparation method of 10mM PBS antibody coating solution: 2.901g of disodium hydrogen phosphate, 0.2964g of sodium dihydrogen phosphate and 8.775g of sodium chloride are placed in a volumetric flask, double distilled water is added, the mixture is shaken gently to be dissolved completely, then the double distilled water is added continuously until the volume is 1000mL, the mixture is mixed evenly, and the pH value is adjusted to 7.4 by concentrated hydrochloric acid.
Example 1, 10mM PBS + 3 wt% trehalose antibody coating preparation method: a clean centrifuge tube was filled with 3g trehalose and 100mL 10mM PBS, 100mL 10mM PBS was prepared as in comparative example 2.
Comparative example 3, 10mM PBS + 0.05 wt% casein antibody coating solution preparation method: 0.05g of casein and 100mL of 10mM PBS were added to a clean centrifuge tube, and 100mL of 10mM PBS was prepared as in comparative example 2.
Comparative example 4, 10mM PBS + 3 wt% BSA antibody coating solution preparation method: 3g BSA and 100mL 10mM PBS were added to a clean centrifuge tube, and 100mL 10mM PBS was prepared as in comparative example 2.
The results of the comparative test using the internal quality control of the enterprise are shown in table 4 below:
TABLE 4 quality control test results (unit: pmol/L) for different antibody coating solutions
Note: the assay buffer was formulated as in example 1, with 0.5 wt% PVP, 0.2 wt% S9, 2 wt% BSA, and 0.5 v/v% PC300 in 20mM Tris solution.
TABLE 5
TABLE 6
TABLE 7
TABLE 8
The above results show that the correlation coefficient of the kit on the market of the example 1 is 0.9985, the correlation coefficient of the comparative example 2 is 0.865, the correlation coefficient of the comparative example 3 is 0.8086, the correlation coefficient of the comparative example 4 is 0.9285, and the best correlation between the example 1 and the kit on the market is obtained.
Example 5
1. Carrying out stability investigation on the assembled dry immunofluorescence quantitative human peptide (CPP) detection kit
The dry immunofluorescence quantitative human Copeptin (CPP) detection kit is placed at 37 ℃ for destructive test, and the test result of the stability of the kit is as follows:
TABLE 9 test results of quality control products after accelerated destruction of kit at 37 deg.C (unit: pmol/L)
The test shows that the kit can be stable for at least 180 days at 37 ℃, and according to the stability experiment principle, the Arrhenius formula: d (Ink)/dT = Ea/RT2 Ea, is stored for 24 months at normal temperature, is equivalent to 180 days of destruction at 37 ℃, can meet the clinical requirements of hospital clinics and health quarantine departments, and can also be used for disease diagnosis research of colleges and universities and scientific research institutions.
2. And (3) sample testing:
the kit and the kit on the market are subjected to sample test, and the results are shown in the following table 7:
TABLE 10 CPP sample alignment results
TABLE 11
The result shows that the correlation coefficient of the test result of the kit and the result of the kit on the market is 0.9886; the correlation is good, and the kit can be used for clinical diagnosis.
The detection data such as the embodiment data, the clinical experiment data, the stability test data, the comparative example test and the like prove that the kit provided by the invention is used for detecting the CPP in the clinical serum specimen, has higher correlation with the detection result of a commercial CPP detection ELISA kit, has better stability, can accurately and quickly detect the content of the peptide-loaded peptide (CPP), and is simple to operate and convenient to use. The use of a fluoroimmunoassay quantitative analyzer greatly shortens the assay time (15 minutes is the result that can be read), while in other methods more than 60 minutes are required. Therefore, our method can provide an advantageous tool for the rapid and accurate detection of CPP in clinical samples.
The invention provides a dry immunofluorescence quantitative human Copeptin (CPP) detection kit, which is marked by fluorescent microspheres, and a fluorescent antibody is immobilized on an independent binding pad, so that the storage temperature requirement of a liquid phase antibody buffer solution is reduced; meanwhile, the types of detection samples are increased, including serum, plasma and whole blood; the invention selects chicken IgY and goat anti-chicken IgY paired antibodies as independent quality control line antibodies, has less cross with the mouse antibody, good specificity and stable quality control line signal value, and is beneficial to improving the accuracy and the repeatability of products; the blocking agent is added into the sample pad to reduce the influence of the interference antibody on the immunity detection to the maximum extent and avoid the false positive or false negative result of the detection.
The above description is only exemplary of the preferred embodiment of the present invention, and should not be taken as limiting the invention, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A dry immunofluorescence quantitative method human Copeptin (CPP) detection kit, characterized in that: including box body (1), upper cover (2) and reagent strip (12), box body (1) and the mutual buckle of upper cover (2) are connected, form between box body (1) and upper cover (2) and hold the chamber, and reagent strip (12) are located and hold the intracavity, reagent strip (12) are including sample pad (3), whole blood filter pad (4), combination pad (5), nitrocellulose membrane (6) and absorbent paper (7), absorbent paper (7) are located the one end of nitrocellulose membrane (6), combination pad (5) are located the other end of nitrocellulose membrane (6), whole blood filter pad (4) are located the one end of keeping away from nitrocellulose membrane (6) of combination pad (5), sample pad (3) are located the one end of keeping away from combination pad (5) of whole blood filter pad (4), the peridium has matter accuse C line (9) in proper order on nitrocellulose membrane (6), A detection T line (8), wherein the quality control C line (9) is arranged at one side close to the absorbent paper (7), and the detection T line (8) is arranged at one side close to the combination pad (5);
the upper cover (2) is provided with a sample adding hole (10) and a detection window (11), the sample adding hole (10) is positioned above the sample pad (3), and the detection window (11) is positioned above the detection T line (8) and the quality control C line (9);
the quality control C line (9) is coated with goat anti-chicken IgY antibody through antibody coating liquid, the concentration is 0.2-4mg/mL, and the antibody coating liquid of the quality control C line (9) is prepared by 10mM PBS containing 0.5-5 wt% trehalose; the detection T line (8) is prepared by coating a mouse anti-human CPP monoclonal antibody with an antibody coating solution, wherein the concentration of the antibody coating solution is 0.5-3mg/mL, and the antibody coating solution of the detection T line (8) is 10mM PBS containing 0.5-5 wt% of sucrose.
2. The dry immunofluorescence quantitative human Copeptin (CPP) detection kit according to claim 1, wherein: the solid phase of the combination pad (5) is provided with a fluorescent antibody, the fluorescent antibody comprises fluorescent microsphere solid phase liquid, a mouse anti-human CPP monoclonal antibody of 0.1-1mg/mL marked by the fluorescent microsphere and chicken IgY of 0.1-1mg/mL, and the fluorescent microsphere solid phase liquid is prepared according to the conditions that 20mM, pH 8.0 and Tris contain 0.01-0.2 wt% of casein, 2-5 wt% of trehalose, 2-10 wt% of sucrose, 0.01-10 wt% of PVP and 0.01-5 wt% of Tween.
3. The dry immunofluorescence quantitative human Copeptin (CPP) detection kit according to claim 2, wherein: the combined pad (5) needs to be pretreated by treatment liquid, and the treatment liquid contains macromolecular polymer and nonionic surfactant which are convenient for the fluorescent microspheres to rapidly release, protein for protecting the activity of the fluorescent microsphere antibody and saccharide for increasing the activity of the labeled antibody.
4. The dry immunofluorescence quantitative human Copeptin (CPP) detection kit according to claim 3, wherein: the treatment fluid is 10mM PBS, the macromolecular polymer is 0.1-10 wt% PVP and 0.01-5 wt% PVA, the nonionic surfactant is 0.01-5 wt% Tween, the protein for protecting the activity of the fluorescent microsphere antibody is 0.01-10 wt% BSA, and the saccharide for increasing the activity of the labeled antibody is 1-10 wt% trehalose.
5. The dry immunofluorescence quantitative human Copeptin (CPP) detection kit according to claim 4, wherein: the method for labeling the fluorescent microspheres comprises the following steps:
the microspheres are subjected to EDC/Sulfo-NHS coupled antibody in a two-step method, the microspheres are washed for 1 time by an activation buffer (50 mM MES buffer PH 6.0) before EDC and Sulfo-NHS are activated, and the microspheres are centrifuged and redissolved by the activation buffer; according to the microsphere: EDC: activating for 20-60min at a sulfoo-NHS mass ratio of 1:0.1-1:0.1-3, centrifuging, re-dissolving with an activation buffer solution, washing for 1 time, centrifuging, re-dissolving with a coupling buffer solution (50 mM MES buffer solution pH 6.5) with coupling of 0.1-1mg antibody per mg microsphere, performing ultrasonic treatment, and mixing and incubating for 2-3h by using a rotary disc mixer; quenching with 100 mM glycine, and incubating for 20-30 min in a rotary disc mixer; centrifuging, washing microspheres for 2 times by using a microsphere sealing buffer solution, centrifuging, re-dissolving by using the microsphere sealing buffer solution, and sealing for 2-4h by using a rotating disc type mixer; centrifuging, re-dissolving the microsphere storage solution, ultrasonically maintaining the microspheres in a monodisperse state, and storing at 4 ℃.
6. The dry immunofluorescence quantitative human Copeptin (CPP) detection kit according to claim 5, wherein: the microsphere blocking buffer solution is prepared according to 20mM and pH 8.0 Tris containing 0.2-5 wt% of casein; the microsphere storage solution is prepared according to 20mM, pH 8.0 and Tris containing 0.2-5 wt% of casein, 1-10 wt% of trehalose, 0.01-5 wt% of Tween and 0.05-0.5 wt% of sodium azide.
7. The dry immunofluorescence quantitative human Copeptin (CPP) detection kit according to claim 6, wherein: the method for the fluorescent antibody solid phase comprises the following steps: diluting the marked fluorescent microspheres to 3-20 times by using fluorescent microsphere solid-phase liquid, spraying the fluorescent microspheres to the pretreated combination pad (5) according to the amount of 2-10uL/cm, and drying in a 37 ℃ oven.
8. The dry immunofluorescence quantitative human Copeptin (CPP) detection kit according to claim 7, wherein: the sample pad (3) needs to be pretreated by treatment fluid, the formula of the treatment fluid is that 10mM PBS contains 0.01-2mg/mL sealant, and the pretreatment method of the sample pad (3) is that the whole glass fiber is placed in the pretreatment fluid, completely soaked for 30min, then placed in a 37 ℃ oven for drying, and stored for standby.
9. The dry immunofluorescence quantitative human Copeptin (CPP) detection kit according to claim 8, wherein: the detection method of the dry immunofluorescence quantitative human Copeptin (CPP) detection kit comprises the following steps: adding 100 mu L of sample to be detected into a sample adding hole (10), horizontally placing for 15 min at room temperature, combining the CPP in the sample to be detected and the mouse anti-human CPP monoclonal antibody marked by the fluorescent microspheres on the binding pad (5) to form an antigen-antibody complex, moving forwards along the nitrocellulose membrane (6) under the action of chromatography, capturing by the mouse anti-human CPP monoclonal envelope antibody to form a complex when moving to a detection T line (8), wherein the fluorescent antibody signal of the complex is in direct proportion to the CPP concentration; the chicken IgY antibody marked by the fluorescent microspheres on the combined pad (5) is combined with the goat anti-chicken IgY antibody to form a compound when continuously migrating to the quality control C line (9); after being analyzed by a fluorescence immunoassay quantitative analyzer, the concentration of CPP in the sample can be calculated.
10. The dry immunofluorescence quantitative human Copeptin (CPP) detection kit according to claim 9, wherein: the manufacturing method of the sample to be detected comprises the following steps: 100 mu L of serum or plasma or whole blood sample is added into a single aliquot of detection buffer and mixed well, wherein the sample detection buffer is 20mM, and the pH is 8.0 Tris and contains 0.01-3 wt% PVP, 0.1-5 wt% S9, 0.1-5 wt% BSA and 0.1-0.5 v/v% PC300 for preparation.
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